Gene regulatory evolution is a major driver for phenotypic divergence. While this has been well-studied in development, its importance in non-developmental processes is far less understood. The overall vision of the lab is to fully characterize the evolutionary rewiring of gene regulatory networks (GRNs) for major stress responses in opportunistic yeast pathogens, and elucidate the contribution of such changes to the survival and virulence in the host. The lab previously discovered a significant difference in how a commensal and opportunistic yeast pathogen, Candida glabrata, regulates its Phosphate Starvation (PHO) response compared with its non-commensal relative, Saccharomyces cerevisiae: the commensal has dramatically expanded its PHO response targets both in number and in function, which was attributed to derived changes in its master transcription factor (TF) by being less dependent on the co-TF. The goal of the lab in the next five years is to determine the genetic and mechanistic bases of this novel mode of TF evolution, i.e. acquiring new targets by reducing co-TF dependence, and the effect of such evolution on stress resistance to phosphate starvation as well as combinatorial stresses in the host. Understanding how this model stress response evolved by itself and in its interaction with other stress responses will begin to elucidate the principles for stress response evolution in commensal yeasts in general. To reach this goal, three Directions will be pursued: 1) Elucidate the mechanisms of a novel mode of TF evolution and its impact on the downstream response, using biophysical and fitness assays; 2) Dissect the crosstalk between stress responses and how it evolved in commensal yeasts, by determining the interaction of PHO response with oxidative stress and general stress responses in C. glabrata and S. cerevisiae; 3) Determine how the PHO network evolved in other commensal yeasts and related non- commensals, using transcriptome profiling coupled with genome-wide Chromatin-IP. Research proposed in this application is innovative because the evolutionary approach will identify key changes in the wiring of the stress response GRNs underlying host adaptation. The proposed research is significant because it will both shed light on the general principles for GRN evolution, and will provide a conceptual framework for developing novel antifungal strategies targeting stress responses.